The present invention relates to novel artificial particles useful as carriers for immunoassay, in particular, particle immunoassay, to preparation thereof and to immunoassay reagent comprising the particles.
The method for particle immunoassay is based on the steps of using particles of appropriate size as carriers, adsorbing antigens or antibodies onto the carriers and observing the agglutination of the thus sensitized carriers caused by antibodies or antigens corresponding thereto.
Examples of the most popular carriers conventionally used in particle immunoassay include erythrocytes of various animals such as sheep and chicken, and artificial synthetic polymer particles such as polystyrene latex particles. Although the former method in which the erythrocytes of animals are used as the carriers has been known as one utilizing so-called hemagglutination, this method has advantages in that it can be applied to many kinds of antigens and antibodies and, for example, the immunoassay can be finished in a short time (1 to 2 hours) by employing the microtiter method. However, there are disadvantages in that the erythrocytes per se have inherent antigenecity so that non-specific agglutination arises easily, and that differences in the properties of the erythrocytes depending on the individual, season and the like are very large and the erythrocytes cannot be obtained in a uniform quality since they are collected from living animals. Furthermore, although there is an advantage in that the size of the erythrocytes is constant for a given kind of animal, this causes a disadvantage in that there cannot be obtained particles having a desired size in accordance with the purpose of use.
On the other hand, synthetic polymer particles such as polystyrene latex generally have a particle diameter of about 0.1.about.1 .mu.m and the particles are particularly useful as carriers for agglutination reaction. The particles have advantages in that the particles per se have no antigenecity and can be constantly obtained in a uniform quality and in a large amount. However, in the case where the microtiter method is used in order to obtain a high sensitivity and to conduct quantitative analysis, the carriers consisting of the conventional synthetic polymer particles have a problem in that a long time period is required for sedimentation, compared with the case of using erythrocytes as carriers and, therefore, the assay cannot be rapidly finished. In addition, there is a possibility of natural agglutination, i.e., non-specific agglutination, occurring in the natural PH region preferably for a immunoreaction medium.
Although it is known that natural inorganic particles such as kaolin and carbon powder are useful as the carriers mentioned above, these particles have disadvantages in that the antibodies or antigens are difficult to sensitize to a high degree and cannot easily be selected in a constant particle range, and therefore, the particles can only be used in an extremely limited field.
Furthermore, there has recently been developed an artificial carrier comprising gelatin, water soluble polysaccharides and sodium polymethacrylate and being crosslinked by an aldehyde crosslinking agent (see Japanese Patent Un-examined Publication Nos. 57-153658 and 57-160465) and this carrier is being put to use as a carrier consisting of gelatin particles instead of animal erythrocytes. The above mentioned Japanese Patent Un-examined Publications disclose that the artificial particles have properties similar to those of animal erythrocytes, which are the best conventional carriers for immunoagglutination reaction, are also chemically and physically stable, have no antigenecity, and can be easily prepared in a desired particle size in a large amount.
However, gelatin is a natural protein material and its properties change depending on the raw material thereof so that the particles cannot always be prepared with uniform properties. Further, the trend toward greater speed and automation in the field of clinical analysis makes it desirable to be able to carry out the reaction in the agglutination reaction analysis more rapidly than possible using animal erythrocytes or gelatin particles.
Therefore, the present invention aims to provide novel carrier particles which are more uniform and stable and enable the agglutination reaction to be carried out more rapidly, compared with the conventional carriers. The carrier particles of the present invention are prepared from a starting material which is not a natural material but a synthetic material, i.e., polyamino acid, where the coacervation method is used as a granulation method.
Contrary to gelatin, the polyamino acid used in the present invention does not gelatinize at room temperature in the granulation process, so that it is possible to prepare coacervate particles. In this method, it is unnecessary to cool the system at the time of making the particles insoluble and also unnecessary to control the temperature of the system, since the speed of the reaction between the particles and the crosslinking agent is slow.
It is suggested in Japanese Patent Un-examined Publication No. 55-94636 that synthetic polyamino acid be used as carrier particles for antigen-antibody reaction. The publication relates to a microcapsule for use in antigen-antibody reaction obtained by the coacervation microcapsule technique and discloses on page 5 line 2 from the bottom that polyamino acid resin is usable as a material capable of forming a capsule shell. However, this prior art substantially discloses gelatin-gum arabic capsule particles, the core of which is formed from oily material and does not specifically disclose any method for preparing capsule particles by using a particular polyamino acid under particular conditions. In fact, experiments conducted by the present inventors revealed that it was very difficult to obtain particles using a commercially available polyamino acid, for example, poly-L-glutamic acid, under coacervation.
Furthermore, Japanese Patent Un-examined Publication No. 62-1728 discloses a polyamino acid spherical particle and a method for preparation thereof and also discloses that one of its uses is as a latex for bioreaction. However, since the particles are prepared by dissolving hydrophobic polyamino acid in an organic solvent and dispersing the thus prepared solution into aqueous medium which is a non-solvent, the thus-prepared particles are essentially hydrophobic and have disadvantages similar to those of polystyrene latex. In addition, as is clear from the Examples set out in the specification, the spherical particles prepared by this conventional method are of a particle diameter of about 40.about.75 .mu.m or 75.about.200 .mu.m and it has been confirmed that even when particles of not more than 10 .mu.m are obtained by sieving the thus obtained particles are not true spheres and do not sufficiently sensitize the antigens and antibodies.